Immunochromatographic or lateral flow assays (LFA) have existed for over twenty years and have proved to be simple and easy to use. This assay technology is characterized by exploiting the migration of a liquid sample by capillary forces through a porous or channeled solid phase so that the analyte enters into successive contact with binding molecules of which at least one is bound to the solid phase. In LFA, unlike non-automated immunoassay systems such as conventional enzyme-linked immunosorbent assay (ELISA), the process of contacting the sample with the individual test components that for example bind the analyte occurs as the fluid phase travels through the solid phase by capillary forces once the sample has been loaded, and is not carried out by the operator.
One of the most common LFA formats involves a sandwich-type assays in which the analyte is bound in between two analyte specific binding partners, Such a so called lateral flow device (LFD) consists of four serial zones of permeable material in fluid communication, illustrated in FIG. 1A. Fluid applied to the sample pad via the application well (a1) flows from the sample pad through a conjugate release pad to a test membrane and to finally an absorbance pad. Positions located closer to the outer end of the sample pad are referred to as being upstream and positions closer to absorbance pad are referred to as being downstream. However, there are a number of different LFA and LFD formats known to prior art that vary in the control of different assay-specific analytical parameters. These include non-linear, multiple path and reverse flow LFDs.
In the most common LFD, the first zone is the sample application zone and comprises a single sample pad (SP; b in FIG. 1A), to which the sample as well as other relevant aqueous solutions are applied via an application well, but there may also be more than one application well over the SP (a1, a2 in FIGS. 1B and 1C) to allow the correct flow of the sample and aqueous solutions to the rest of the LFD. The sample application zone can condition the sample, for example by filtering out particulate matter or cells and/or add buffering agents to adjust the pH of the sample, while also ensuring a controlled flow of liquid sample to the rest of the device.
The second zone, which is hereinafter referred to as the conjugate release pad (CRP; c in FIG. 1A), is the next zone to which the sample migrates. In its most common form the CRP contains an analyte-specific binding partner conjugated to a detection moiety, this conjugate hereinafter being referred to as the analyte-specific detection conjugate (ASDC). The ASDC is contained in dried form within the CRP and is dissolved in and migrates with the sample once it comes into contact with it. In sandwich-type assays, the analyte-specific agent is a binding partner which specifically binds to analyte in the sample to form a complex.
The third zone, which is hereinafter referred to as the test zone (d in FIG. 1A), is the next zone contacted by the sample. The test zone usually consists of a nitrocellulose membrane and contains two narrow bands of agent bound to the solid phase and placed at a right angle to the direction of sample flow. The first band, situated closest to the SP and hereinafter referred to as the test band (e in FIG. 1A), consists of another analyte-specific agent, which is immobilized at the site and is hereinafter referred to as the analyte-specific capture agent (ASCA). In sandwich-type assays the ASCA is a binding partner which specifically binds the analyte. When the sample migrates to and through the test band the ASCA binds both free and any ASDC-complexed analyte present, causing an accumulation of both at the test band. If sufficient analyte is present in the sample and if a sufficient proportion is bound to the ASDC, the accumulation of the ASDC at the test band is detected. The second band (f in FIG. 1A) is usually a control band placed downstream to the test band, and may simply consist of an immobilized binding partner specific to the ASDC but not to the analyte. Sufficient build-up of ASDC at the control line indicates the correct passage of the sample though the LFD.
The fourth and final zone with which the sample comes into contact is the absorbance pad (g in FIG. 1A) consisting of an absorbent material which acts to receive the residual sample after it has passed over the test zone, and thus allowing the majority of the sample applied to the LFD to pass through the test zone.
Another LFA format is a competitive-type assay. In competitive-type assays either the ASDC or the ASCA contains the analyte or a derivative of the same instead of an analyte specific binding partner such as is present in sandwich-type assays. In competitive-type assays, analyte present in the sample has a negative influence on the accumulation of ASDC at the test band.
The following description will focus on the phenomena involved with sandwich-type assay formats but the same or similar phenomena also occur in competitive-type assays with changes in the agent to which the phenomena relate. In LFAs the band intensity represents the number of ASDC molecules or particles accumulated at the test band. This number relates to the number of analyte molecules reacted with both the ASCA and ASDC. In order to quantify the analyte in a test sample, the band intensity is compared to that obtained from samples of known analyte content. In a standard LFA, the coefficient of variation is around 20-25% even for a fully optimized assay. An important source of error associated with LFAs is run-to-run variation in the volume of sample exposed to i) the ASDC, and ii) the ASCA, or a combination of the two. These two sources of error will be treated separately in the following, as they result from separate mechanisms and the problems of reducing the variation in each require separate solutions.
i) Variation Associated with Contact between the Analyte and the ASDC
In standard LFAs, the sample is applied to the LFD and migrates from the SP (b in FIG. 1) to the CRP (c in FIG. 1), where it starts to dissolve the ASDC. Once dissolved, the ASDC migrates with the sample to the test zone, at the same time contacting and binding the analyte. Contact between the ASDC and the analyte in the sample depends on the rate of dissolution of ASDC by the sample and the total volume of sample that passes through the CRP. Ideally, the ASDC should be released into the solution evenly over the total sample volume, allowing it to come into contact with all of the analyte distributed in the total sample volume or at least in a constant manner so that the volume of sample contacted is constant. Unfortunately the release rate of ASDC from the CRP can be affected by sample-to-sample and environmental variations. Parameters such as temperature, sample viscosity, sample pH and the ionic strength of the sample may all affect the release of the ASDC from the CRP and therefore the volume of sample contacted.
European patent document EP1716420 and international patent document WO 2006/083367 teach quantitative LFA methods in which the detection conjugate (ASDC) is combined with the sample before it is applied to the LFA device. In this way the conjugate interacts evenly with all of the analyte in the sample, thus overcoming the effect of variation in the CRP release rate.
Multiple-step methods have also been developed in which the ASDC enters into contact with the test band after the analyte present in the sample has been immobilized at the test band by the ASCA. The amount of analyte immobilized relates to the amount of analyte that has entered into contact with the ASCA. The ASDC then enters into contact with the immobilized analyte. Ideally an excess of ASDC contacts with the immobilized analyte, binding to most if not all of the immobilized analyte, so that the response obtained is not affected by the normal test-to-test variations in the amount of ASDC that contacts with the immobilized analyte.
ii) Variation Associated with Contact between the Analyte and the ASCA
The final response in a standard LFA relates to the number analyte molecules that are bound to the ASCA and therefore depends on the amount of sample that comes in contact with the ASCA. Variations in the flow characteristics of the sample through the LFD can affect both the total volume of sample passing through the test band and the proportion of analyte molecules in the sample that contact the ASCA immobilized in the test band. If the sample flow is slower, the analyte molecules have a longer time to diffuse to and interact with the immobilized ASCA molecules as they pass through the test band. This means that the ASCA is able to interact with a higher proportion of the analyte molecules in the sample. However, as the flow rate increases, more sample and therefore more analyte can pass though the test band contacting the ASCA. Factors such as temperature affect both the flow rate of the sample and the diffusion rate of the analyte. Other factors such as sample viscosity and variations in the membrane properties, for example due to differences in the humidity of the atmosphere in which the LFA is run, will all affect on one or both of these properties. As a result it is difficult to predict the effects of between-sample variation and the effects of variation in environmental conditions.
International patent document WO 2006/083367 teaches methods in which errors due to variations in the volume of sample that enters into contact with the ASCA are compensated for by using analyte-independent binding pairs that are not endogenous to the sample, with one of the pair being immobilized at the control band. Variation in the volume of sample contacting the ASCA immobilized at the test band are mirrored in the control band, the signal from which can then be used to correct the reading obtained from the test band.